Biotechnology and genetic engineering pbio 4500 5500
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Biotechnology and Genetic Engineering PBIO 4500/5500. Characterization of DNA clones including: Restriction Enzyme (RE) mapping Subcloning Southerns Northerns* Westerns Hybrid-select translation* DNA sequencing* PCR. Characterization: RE mapping.

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Biotechnology and Genetic Engineering PBIO 4500/5500

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Biotechnology and genetic engineering pbio 4500 5500

Biotechnology and Genetic EngineeringPBIO 4500/5500

Characterization of DNA clones including:

Restriction Enzyme (RE) mapping

Subcloning

Southerns

Northerns*

Westerns

Hybrid-select translation*

DNA sequencing*

PCR


Characterization re mapping

Characterization: RE mapping

Predict what would happen with a double digest.


Characterization subcloning

Characterization: Subcloning

  • Refers to the process of cloning smaller pieces of a large DNA cloning into another cloning vector

  • E.g., subcloning the individual EcoRI fragments of a partial EcoRI lambda genomic clone into plasmid vectors

  • Facilitates amplication and analysis of the subcloned DNA, including probe preparation


Characterization southern blot hybridization

Characterization: Southern blot hybridization

-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)

-developed by Edwin Southern


Characterization northern blot hybridization

Characterization: Northern blot hybridization

X RNA

X

x

salt

X

RNA

-transfer of RNA from a gel to a membrane (e.g., nitrocellulose, nylon)

-reveals mRNA size (and approximate protein size), tissue- and organ-

specific expression, and kinetic patterns of expression


Biotechnology and genetic engineering pbio 4500 5500

Experimental Figure 5.27 Northern blot analysis reveals increased expression of b-globin mRNA in differentiated erythroleukemia cells.


Biotechnology and genetic engineering pbio 4500 5500

Experimental Figure 5.28 In situ hybridization can detect activity of specific genes in whole and sectioned embryos.


Characterization western blotting

Characterization: Western blotting

X Protein

Enzyme

reaction

or

X

React with

Antibody

X

x

Buffer; requires electric current

X

-transfer of protein from a gel to a membrane (e.g., nitrocellulose, nylon)

-requires the use of an electric current to facilitate transfer


Animations for western blotting

Animations for Western Blotting

  • SDS gel electrophoresis- MCB Chapter 3

  • http://bcs.whfreeman.com/lodish7e/#800911__812035__

  • Western blotting or Immunoblotting- MCB Chapter 3

  • http://bcs.whfreeman.com/lodish7e/#800911__812034__


Characterization hybrid select translation

Characterization: Hybrid-select translation


Characterization dna sequencing

Characterization: DNA Sequencing

The dideoxynucleotide structure (note 3’H) is the key to dideoxy DNA sequencing


Dideoxy dna sequencing animation mcb chapter 5

Dideoxy DNA Sequencing Animation- MCB Chapter 5

  • http://bcs.whfreeman.com/lodish7e/#800911__812048__


Characterization dna sequencing1

Characterization: DNA sequencing


Dna sequencing

DNA sequencing


Biotechnology and genetic engineering pbio 4500 5500

Automated

DNA sequencing

Capillary electrophoresis


Biotechnology and genetic engineering pbio 4500 5500

Pyrosequencing http://www.biotagebio.com/DynPage.aspx?id=7454


454 sequencing massively parallel pyrosequencing

454 Sequencing (massively parallel pyrosequencing)

How it Works

System Workflow: One Fragment = One Bead = One Read

The complete sequencing workflow of the Genome Sequencer FLX System comprises four main steps, leading from purified DNA to analyzed results. These basic steps include: 1) Generation of a single-stranded template DNA library, 2)Emulsion-based clonal amplification of the library, 3) Data generation via sequencing-by-synthesis, and 4) Data analysis using different bioinformatics tools

Sample Input and Fragmentation

The Genome Sequencer FLX System supports the sequencing of samples from a wide variety of starting materials including genomic DNA, PCR products, BACs, and cDNA. Samples such as genomic DNA and BACs are fractionated into small, 300- to 800-basepair fragments. For smaller samples, such as small non-coding RNA or PCR amplicons, fragmentation is not required. Instead, short PCR products amplified using Genome Sequencer fusion primers can be used for immobilization onto DNA capture beads as shown below under "One Fragment = One Bead".

Library Preparation

Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - are added to each fragment. The adaptors are used for purification, amplification, and sequencing steps. Single-stranded fragments with A and B adaptors compose the sample library used for subsequent workflow steps.

One Fragment = One Bead

The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Each bead carries a unique single-stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture resulting in microreactors containing just one bead with one unique sample-library fragment.

emPCR (Emulsion PCR) Amplification

Each unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this results in a copy number of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads.

One Bead = One Read

The clonally amplified fragments are enriched and loaded onto a PicoTiterPlate device for sequencing. The diameter of the PicoTiterPlate wells allows for only one bead per well. After addition of sequencing enzymes, the fluidics subsystem of the Genome Sequencer FLX Instrument flows individual nucleotides in a fixed order across the hundreds of thousands of wells containing one bead each. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera of the Genome Sequencer FLX Instrument. For a detailed explanation of this reaction see Sequencing Chemistry.

Data Analysis

The combination of signal intensity and positional information generated across the PicoTiterPlate device allows the software to determine the sequence of more than 1,000,000 individual reads per 10-hour instrument run simultaneously. For sequencing-data analysis, three different bioinformatics tools are available supporting the following applications: de novo assembly up to 400 megabases; resequencing genomes of any size; and amplicon variant detection by comparison with a known reference sequence.

See http://www.454.com/products-solutions/how-it-works/index.asp


Pcr animation mcb chapter 5

PCR Animation- MCB Chapter 5

  • http://bcs.whfreeman.com/lodish7e/#800911__812049__


The polymerase chain reaction pcr

The Polymerase Chain Reaction (PCR)

  • PCR is a cloning method without a host

  • Thermus aquaticus, a hot spring bacterium, produces Taq polymerase

  • Taq polymerase, unlike E. coli DNA polymerase, is not denatured at 95°C


Chemical synthesis of dna oligonucleotide synthesis using phosphoramidite chemistry

Chemical synthesis of DNA(oligonucleotide synthesis using phosphoramidite chemistry)


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